SummaryMembers of the SNF1-related protein kinase-1 (SnRK1) subfamily of protein kinases are higher plant homologues of mammalian AMP-activated and yeast SNF1 protein kinases. Based on analogies with the mammalian system, we surmised that the SnRK1 kinases would be regulated by phosphorylation on a threonine [equivalent to Thr175 in Arabidopsis thaliana SnRK1 (AKIN10)] within the`T loop' between the conserved DFG and APE motifs. We have raised an antibody against a phosphopeptide based on this sequence, and used it to show that inactivation of two spinach SnRK1 kinases by protein phosphatases, and reactivation by a mammalian upstream protein kinase, is associated with changes in the phosphorylation state of this threonine. We also show that dephosphorylation of this threonine by protein phosphatases, and consequent inactivation, is inhibited by low concentrations of 5¢-AMP, via binding to the substrate (i.e. the kinase). This is the ®rst report showing that the plant SnRK1 kinases are regulated by AMP in a manner similar to their mammalian counterparts. The possible physiological signi®cance of these ®ndings is discussed.

ATP-sensitive K(+) (K(ATP)) channels in the heart are normally closed by high intracellular ATP, but are activated during ischemia to promote cellular survival. These channels are heteromultimers composed of Kir6.2 subunit, an inwardly rectifying K(+) channel core, and SUR2A, a regulatory subunit implicated in ligand-dependent regulation of channel gating. Here, we have shown that the muscle form (M-LDH), but not heart form (H-LDH), of lactate dehydrogenase is directly physically associated with the sarcolemmal K(ATP) channel by interacting with the Kir6.2 subunit via its N-terminus and with the SUR2A subunit via its C-terminus. The species of LDH bound to the channel regulated the channel activity despite millimolar concentration of intracellular ATP. The presence of M-LDH in the channel protein complex was required for opening of K(ATP) channels during ischemia and ischemia-resistant cellular phenotype. We conclude that M-LDH is an integral part of the sarcolemmal K(ATP) channel protein complex in vivo, where, by virtue of its catalytic activity, it couples the metabolic status of the cell with the K(ATP) channels activity that is essential for cell protection against ischemia.

Cardiac sarcolemmal ATP-sensitive K + (K ATP ) channels, composed of Kir6.2 and SUR2A subunits, couple the metabolic status of cells with the membrane excitability. Based on previous functional studies, we have hypothesized that creatine kinase (CK) may be a part of the sarcolemmal K ATP channel protein complex. The inside-out and whole cell patch clamp electrophysiology applied on guinea pig cardiomyocytes showed that substrates of CK regulate K ATP channels activity. Following immunoprecipitation of guinea-pig cardiac membrane fraction with the anti-SUR2 antibody, Coomassie blue staining revealed, besides Kir6.2 and SUR2A, a polypeptide at ∼48 kDa. Western blotting analysis confirmed the nature of putative Kir6.2 and SUR2A, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis identified p48 kDa as a muscle form of CK. In addition, the CK activity was found in the anti-SUR2A immunoprecipitate and the cross reactivity between an anti-CK antibody and the anti-SUR2A immunoprecipitate was observed as well as vice verse. Further results obtained at the level of recombinant channel subunits demonstrated that CK is directly physically associated with the SUR2A, but not the Kir6.2, subunit. All together, these results suggest that the CK is associated with SUR2A subunit in vivo, which is an integral part of the sarcolemmal K ATP channel protein complex. Keywordsheart; K ATP channels; SUR2A; Kir6.2 In the heart, creatine kinase (CK) is a major phosphotransfer system essential in supporting cardiac energy balance (1). To ensure communications between sites that generate, use, and sense ATP, cardiac cells rely on phosphotransfer networks that facilitate the transfer and distribution of energy-rich phosphoryls between cellular compartments in a kinetically and thermodynamically efficient manner mediated by CK (2).ATP-sensitive K + (K ATP ) channels belong to a group of intracellular ATP sensors and they couple the metabolic status of cell with membrane excitability (3). Numerous studies have demonstrated that potassium channel openers, drugs that promote opening of K ATP channels, decrease infarct size, mimic ischemic preconditioning, and improve functional and energetic recovery of cardiac muscle following ischemic and hypoxic insults (4,5). More recently, evidence has suggested that activation of both sarcolemmal and mitochondrial K ATP channels may promote cellular survival (4), which would agree with the idea that these channels may communicate through phosphotransfer reactions from an intracellular compartment to the cell UKPMC Funders Group Author ManuscriptUKPMC Funders Group Author Manuscript membrane (1). The structure of mitochondrial K ATP channels is still unknown, but the proteins constituting the sarcolemmal K ATP channel complex have been cloned recently (6-8). Sarcolemmal K ATP channels are heteromultimers composed of two structurally distinct proteins (Kir6.2 and SUR2A) (7). The Kir6.2 subunit was shown to form the inwardly rectifying K + channel core, p...